The present invention relates generally to the fields of organic chemistry, biochemistry and/or biological treatment systems. In particular, the present invention is directed to compounds that are capable of releasing nitric oxide (NO) in a controlled manner, as well as methods for the preparation and use thereof.
Nitric oxide has been implicated in a variety of important bioregulatory processes, including neurotransmission, anticoagulation and vasodilation. In addition, NO is an effector molecule released by macrophages and other cells after immunological activation.
NO is synthesized from the amino acid L-arginine by an enzyme, NO synthase. It is believed that there are at least two forms of the enzyme: a constitutive form which releases NO for short periods in response to receptor or physical stimulation, and a second form which is induced after activation of macrophages, endothelial cells and certain other cells by cytokines and which, once expressed, synthesizes NO for extended periods.
The constitutive form of NO synthase is implicated in the transduction mechanism for soluble guanylate cyclase, and thus is involved in one of the mechanisms whereby cells regulate their own function or communicate with others. In addition, the release of NO in the cardiovascular system acts as a general adaptive mechanism whereby the vascular endothelium responds to changes in its environment and regulates blood flow and blood pressure through action on vascular smooth muscle. NO also regulates the interaction between the endothelium and the platelets; it may also play a role in the control of vascular smooth muscle proliferation. The NO released by the constitutive enzyme may also play regulatory roles in other cells; for example, it is known to be linked to the stimulation by the excitatory amino acids of specific receptors in the central nervous system, and it may participate in regulation of the secretion or action of various hormones.
NO released after immunological stimulation by the other form of the enzyme as part of the host defense mechanism has been shown to be cytotoxic or cytostatic for tumor cells and invasive organisms. Further, some forms of local or systemic tissue damage associated with immunological conditions could prove to be related as well to the release of NO. NO may also play a role in the normal regulation of the response of cells to mitogens, or contribute to the cytotoxic actions of other cells that play a role in specific immunity.
NO is itself extremely poisonous and reactive in the presence of oxygen. It is a highly reactive gas in its pure form, attacking metals and plastics, and can only be obtained in relatively low pressure cylinders. Moreover, NO has limited solubility in aqueous media, making it difficult to introduce reliably into most biological systems without premature decomposition.
In view of the central importance of NO as both a transducer and as an effector molecule, however, it is apparent that agents for the controlled release of NO would be invaluable in the ongoing research on the roles of NO in human physiology and pathology. Moreover, the therapeutic potential of agents that may be employed to release NO in a controlled manner is enormous.
Various vasodilators are known which release NO either spontaneously or upon activation by chemical or enzymatic means. For example, the release of NO from molsidomine (N-ethoxycarbonyl-3-morpholinosydnonimine) begins with an enzymatic hydrolysis catalyzed mainly by liver esterases, followed by a pH-dependent ring opening reaction catalyzed by hydroxyl ions; a subsequent oxidative process is then essential for further decomposition and NO release [Bohn, H. and K. Schoenafinger, “Oxygen and Oxidation Promote the Release of Nitric Oxide from Sydnonimines,” J. Cardiovasc. Pharmacol. 14 (Suppl.11):S6-S12 (1989)]. The vasodilators glyceryl trinitrate and sodium nitroprusside release nitric oxide upon redox activation. Other agents, such as iron-sulfur cluster nitrosyls, decompose spontaneously to release NO [Flitney, F. W. et al., “Selective retention of iron-sulphur cluster nitrosyls in endothelial cells of rat isolated tail artery: association with protracted vasodilator responses, J. Physiol. 459:89P (1993)].
One example of a compound that spontaneously releases NO is a class of diazeniumdiolate compounds. Anions such as 1-(N,N-dialkylamino)diazen-1-ium-1,2-diolates (see compound 1 (X=NR2, R=alkyl)) are stable as solid salts, but release up to two moles of NO when dissolved in aqueous solution at physiologically relevant conditions. Compound 1 is shown below in its anionic form. Compound 1 appears in this state throughout the description, the sodium ion is not shown. Keefer et al. have shown that the rate of NO release from diazeniumdiolates is dependent on the nature of the organic anion (X−), and have developed diazeniumdiolate compounds with half-lives of between 1.8 seconds to 56 hours in aqueous buffer at a pH of 7.4 at 37° C. Maragos, C. M.; Morley, D.; Wink, D. A.; Dunams, T. M.; Saavedra, J. E.; Hoffman, A.; Bove, A. A.; Isaac, L.; Hrabie, J. A.; Keefer, L. K. J. Med. Chem. 1991, 34, 3242–3247. (b) Keefer, L. K.; Nims, R. W.; Davies, K. M.; Wink, D. A. Methods in Enzymology 1996, 268, 281–293.

Diazeniumdiolates have shown great potential in a variety of medical applications requiring the rapid production (e.g., to produce a fast, but transient drop in blood pressure) or gradual release of NO (e.g., to study the effects of prolonged cytostasis on vascular smooth muscle cells).
One such strategy proposes to deliver NO to a specific site by anchoring diazeniumdiolate in polymeric matrices, restricting the release of NO to only those cells to which the polymer is in physical contact. Another strategy proposes using pro-drug derivatives that are unable to release NO until they have been metabolically converted to diazenimdiolate by enzymes specific to the target cell type. For example, diazeniumdiolates masked by O2-alkylation were shown to protect the liver in rats from cell death while minimally affecting systemic blood pressure, after being selectively dealkylated by oxidative enzymes specifically found in the liver.
It is therefore an object of the present invention to provide compositions for controlled delivery of nitric oxide that remain stable until it is desired to release NO by a particular triggering means, as well as towards methods for the preparation and uses thereof.
A wide variety of groups labile to photolysis referred to as “caging” structures are known in the art. The commonly employed 2-nitrobenzyl photosensitive protecting group has been used to develop potential photochemical precursors (compound 2, R=H, OCH3, OCH2CO2Et) to diazeniumdiolates. These compounds of Makings and Tsien are described in U.S. Pat. No. 5,374,710 and have the general formula:

Photolysis of compound 2 results in poor yields of NO with correspondingly low quantum yields. Nonetheless, these phototriggered NO donors were used to inhibit thrombin-stimulated platelet aggregation, to study the induction of long-term depression in the cerebellum and to examine long-term potentiation in culture of hippocampal neurons.
However, in the literature, it has been determined that when photolyzed, simple O2-substituted diazeniumdiolates (such as compound 2) have been found to breakdown by at least two primary pathways upon exposure to light [Srinivasan, A.; Kebede, N.; Saavedra, J. E.; Nikolaitchik, A. V.; Brady, D. A.; Yourd E.; Davies, K. M.; Keefer, L. K. and Toscano, J. P. J. Am. Chem. Soc. 2001 123, 5465–5472]. As shown in the reaction pathway below (scheme 1) (and as further described in Example 9 below), the major breakdown pathway of the diazeniumdiolate compounds of Makings and Tsien, involves the formation of potentially carcinogenic nitrosamine (R2NN═O) (compound 12) and a reactive oxygen-substituted nitrene (RON) (compound 13) as by-products (Path A) (shown in scheme 1 below). Minor amounts of NO, potentially produced by secondary photolysis of the nitrosamine are observed. Due to the toxic nature of these breakdown products, pharmaceuticals based on such diazeniumdiolate derivatives should be avoided.

It is one aspect of this invention to avoid the production of toxic byproducts produced by Path A. The prevention of toxic by-products may be achieved by choosing alternate caging structures that help favor the formation of non-toxic by-products upon photolysis.